Jan, Yi-HuaHeck, Diane
E.Casillas, Robert P.Laskin, Debra L.Laskin, Jeffrey D.Thioredoxin Cross-Linking
by Nitrogen Mustard in Lung
Epithelial Cells: Formation of Multimeric Thioredoxin/Thioredoxin
Reductase Complexes and Inhibition of Disulfide ReductionThe thioredoxin (Trx) system, which
consists of Trx and thioredoxin
reductase (TrxR), is a major cellular disulfide reduction system important
in antioxidant defense. TrxR is a target of mechlorethamine (methylbis­(2-chloroethyl)­amine;
HN2), a bifunctional alkylating agent that covalently binds to selenocysteine/cysteine
residues in the redox centers of the enzyme, leading to inactivation
and toxicity. Mammalian Trx contains two catalytic cysteines; herein,
we determined if HN2 also targets Trx. HN2 caused a time- and concentration-dependent
inhibition of purified Trx and Trx in A549 lung epithelial cells.
Three Trx cross-linked protein complexes were identified in both cytosolic
and nuclear fractions of HN2-treated cells. LC-MS/MS of these complexes
identified both Trx and TrxR, indicating that HN2 cross-linked TrxR
and Trx. This is supported by our findings of a significant decrease
of Trx/TrxR complexes in cytosolic TrxR knockdown cells after HN2
treatment. Using purified recombinant enzymes, the formation of protein
cross-links and enzyme inhibition were found to be redox status-dependent;
reduced Trx was more sensitive to HN2 inactivation than the oxidized
enzyme, and Trx/TrxR cross-links were only observed using reduced
enzyme. These data suggest that HN2 directly targets catalytic cysteine
residues in Trx resulting in enzyme inactivation and protein complex
formation. LC-MS/MS confirmed that HN2 directly alkylated cysteine
residues on Trx, including Cys32 and Cys35 in the redox center of
the enzyme. Inhibition of the Trx system by HN2 can disrupt cellular
thiol–disulfide balance, contributing to vesicant-induced lung
toxicity.enzyme;bifunctional alkylating agent;HN 2;Disulfide ReductionThe thioredoxin;549 lung epithelial cells;Lung Epithelial Cells;alkylated cysteine residues;HN 2 treatment;cytosolic TrxR knockdown cells;disulfide reduction system;HN 2 inactivation2015-11-16